PROJECT SUMMARY/ABSTRACT Breakthroughs in our knowledge of the molecular and cellular mechanisms regulating metastasis have yet to be broadly translated into improved survival rates for patients with metastatic disease. Lymph node metastasis in a cancer patient brings with it a poorer prognosis and the recommendation for systemic therapy. However, it is unclear whether lymphatic metastases are only predictive or whether they play a role in cancer progression and the emergence of distant metastases. Evidence shows that treating lymph node metastases improves survival in some patients. The work proposed here aims to build a biological understanding of lymph node metastases in order to define their role in disease progression and identify new therapeutic interventions to improve patient survival. Recently, we have shown that lymph node metastases do not require angiogenesis to grow and do not respond to anti-angiogenic therapy. We will build upon our unique expertise by addressing why lymph node metastasis are such strong prognostic indicators and explore how lymph node metastasis drive cancer progression. Specifically, we will test the hypotheses that lymph node metastases disseminate to distant sites (Aim 1) and inhibit the ability of the immune system to develop and maintain anti-tumor immunity (Aim 2). We will accomplish our goals using innovative animal models that allow state-of-the-art intravital microscopy of spontaneous lymph node metastasis. In Aim 1, we will determine whether cancer cells utilize the fibroblastic reticular cell-lined conduit system in lymph nodes to home to blood vessels and spread to distant sites. We will attempt to block this process. In Aim 2, we will determine whether metastatic lymph nodes retain the ability to initiate immune responses to new antigens. Further, we will determine the mechanism of immune suppression in metastatic lymph nodes. We will attempt to increase immune effector function in lymph node metastases to eradicate lymph node disease and stimulate systemic anti-tumor immunity. In completing these aims, we will have better lymph node focused therapeutic options to limit the growth and spread of cancer from lymph nodes and thus provide better outcomes for patients. Critical elements to achieving these goals are intravital imaging equipment, complex animal models and functional immunological assays, all available in the PI?s laboratory. In addition, the assembled team has complementary expertise that will enable them to overcome any problems that occur during the project. Dr. Padera is an expert in intravital microscopy of the lymphatic system and mechanisms of lymph node metastasis. Dr. Carroll is an immunologist with deep knowledge of fluid and cellular transport in lymph nodes as well as adaptive immunity. Dr. Munn has expertise in image processing and quantitative biology with experience in measuring directed cell migration. Each team member, along with our collaborators, will play a critical role in this interdisciplinary research, uniquely positioning us to address these clinically driven questions. With the completion of our studies we will be able to better contain and eradicate lymph node metastasis to extend survival for patients with metastatic cancer.